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International audience; Although structural studies on the ligand-binding domain (LBD) have established the general mode of nuclear receptor (NR)/coactivator interaction, determinants of binding specificity are only partially understood. The LBD of estrogen receptor-alpha (ERalpha), for example, interacts only with a region of peroxisome proliferator-activated receptor coactivator (PGC)-1alpha, which contains the canonical LXXLL motif (NR box2), whereas the LBD of estrogen-related receptor-alpha (ERRalpha) also binds efficiently an untypical, LXXYL-containing region (NR box3) of PGC-1alpha. Surprisingly, in a previous structural study, the ERalpha LBD has been observed to bind NR box3 of transcriptional intermediary factor (TIF)-2 untypically via LXXYL, whereas the ERRalpha LBD binds this region of TIF-2 only poorly. Here we present a new crystal structure of the ERRalpha LBD in complex with a PGC-1alpha box3 peptide. In this structure, residues N-terminal of the PGC-1alpha LXXYL motif formed contacts with helix 4, the loop connecting helices 8 and 9, and with the C terminus of the ERRalpha LBD. Interaction studies using wild-type and mutant PGC-1alpha and ERRalpha showed that these contacts are functionally relevant and are required for efficient ERRalpha/PGC-1alpha interaction. Furthermore, a structure comparison between ERRalpha and ERalpha and mutation analyses provided evidence that the helix 8-9 loop, which differs significantly in both nuclear receptors, is a major determinant of coactivator binding specificity. Finally, our results revealed that in ERRalpha the helix 8-9 loop allosterically links the LBD homodimer interface with the coactivator cleft, thus providing a plausible explanation for distinct PGC-1alpha binding to ERRalpha monomers and homodimers.

The insect steroid hormone 20-hydroxyecdysone (20E) binds to its cognate nuclear receptor composed of the ecdysone receptor (EcR) and Ultraspiracle (USP) and triggers the main developmental transitions, in particular molting and metamorphosis. We present the crystal structure of the ligand-binding domains of EcR/USP in complex with 20E at 2.4A resolution and compare it with published structures of EcR/USP bound to ponasterone A (ponA). ponA is essentially identical to 20E but lacks the 25-OH group of 20E. The structure of 20E-bound EcR indicates that an additional hydrogen bond is formed compared with the ponA-bound receptor, yet, paradoxically, ponA has a significantly higher affinity for EcR than 20E. Theoretical studies based on docking and free energy methods lead to a rationale for understanding the difference in binding affinities between 20E and ponA. Results of the calculations indicate that the favorable contribution from the extra H-bond made by 25-OH of 20E is counterbalanced by its larger desolvation cost compared with that of ponA. The contribution of 25-OH to the binding affinity is further compared with those of 20- and 22-OH groups. Ligands that lack the 20- or 22-OH group are indeed known to bind less favorably to EcR than 20E, an effect opposite to that observed for ponA. The results indicate that their respective contributions to receptor-ligand complex stability reside mostly in their different contributions to solvation/desolvation. Together, the data demonstrate the critical role of ligand desolvation in determining binding affinity, with general implications for the binding of hormones to their cognate nuclear receptors.

International audience; NGFI-B is a ligand-independent orphan nuclear receptor of the NR4A subfamily that displays important functional differences with its homolog Nurr1. In particular, the NGFI-B ligand-binding domain (LBD) exhibits only modest activity in cell lines in which the Nurr1 LBD strongly activates transcription. To gain insight into the structural basis for the distinct activation potentials, we determined the crystal structure of the NGFI-B LBD at 2.4-angstroms resolution. Superimposition with the Nurr1 LBD revealed a significant shift of the position of helix 12, potentially caused by conservative amino acids exchanges in helix 3 or helix 12. Replacement of the helix 11-12 region of Nurr1 with that of NGFI-B dramatically reduces the transcriptional activity of the Nurr1 LBD. Similarly, mutation of Met414 in helix 3 to leucine or of Leu591 in helix 12 to isoleucine (the corresponding residues found in NGFI-B) significantly affects Nurr1 transactivation. In comparison, swapping the helix 11-12 region of Nurr1 into NGFI-B results in a modest increase of activity. These observations reveal a high sensitivity of LBD activity to changes that influence helix 12 positioning. Furthermore, mutation of hydrophobic surface residues in the helix 11-12 region (outside the canonical co-activator surface constituted by helices 3, 4, and 12) severely affects Nurr1 transactivation. Together, our data suggest that a novel co-regulator surface that includes helix 11 and a specifically positioned helix 12 determine the cell type-dependent activities of the NGFI-B and the Nurr1 LBD.

The estrogen-related receptor (ERR) gamma behaves as a constitutive activator of transcription. Although no natural ligand is known, ERRgamma is deactivated by the estrogen receptor (ER) agonist diethylstilbestrol and the selective ER modulator 4-hydroxytamoxifen but does not significantly respond to estradiol or raloxifene. Here we report the crystal structures of the ERRgamma ligand binding domain (LBD) complexed with diethylstilbestrol or 4-hydroxytamoxifen. Antagonist binding to ERRgamma results in a rotation of the side chain of Phe-435 that partially fills the cavity of the apoLBD. The new rotamer of Phe-435 displaces the "activation helix" (helix 12) from the agonist position observed in the absence of ligand. In contrast to the complexes of the ERalpha LBD with 4-hydroxytamoxifen or raloxifene, helix 12 of antagonist-bound ERRgamma does not occupy the coactivator groove but appears to be completely dissociated from the LBD body. Comparison of the ligand-bound LBDs of ERRgamma and ERalpha reveals small but significant differences in the architecture of the ligand binding pockets that result in a slightly shifted binding position of diethylstilbestrol and a small rotation of 4-hydroxytamoxifen in the cavity of ERRgamma relative to ERalpha. Our results provide detailed molecular insight into the conformational changes occurring upon binding of synthetic antagonists to the constitutive orphan receptor ERRgamma and reveal structural differences with ERs that explain why ERRgamma does not bind estradiol or raloxifene and will help to design new selective antagonists.

The heterotrimeric transcription factor NF-Y recognizes with high specificity and affinity the CCAAT regulatory element that is widely represented in promoters and enhancer regions. The CCAAT box acts in concert with neighboring elements, and its bending by NF-Y is thought to be a major mechanism required for transcription activation. We have solved the structure of the NF-YC/NF-YB subcomplex of NF-Y, which shows that the core domains of both proteins interact through histone fold motifs. This histone-like pair is closely related to the H2A/H2B and NC2alpha/NC2beta families, with features that are both common to this class of proteins and unique to NF-Y. The structure together with the modeling of the nonspecific interaction of NF-YC/NF-YB with DNA and the full NF-Y/CCAAT box complex highlight important structural features that account for different and possibly similar biological functions of the transcriptional regulators NF-Y and NC2. In particular, it emphasizes the role of the newly described alphaC helix of NF-YC, which is both important for NF-Y trimerization and a target for regulatory proteins, such as MYC and p53.

International audience; To further our understanding of the transcription/DNA repair factor TFIIH, we investigated the role of its p52 subunit in TFIIH function. Using a completely reconstituted in vitro transcription or nucleotide excision repair (NER) system, we show that deletion of the C-terminal region of p52 results in a dramatic reduction of TFIIH NER and transcription activities. This mutation prevents promoter opening and has no effect on the other enzymatic activities of TFIIH. Moreover, we demonstrate that intact p52 is needed to anchor the XPB helicase within TFIIH, providing an explanation for the transcription and NER defects observed with the mutant p52. We show that these two subunits physically interact and map domains involved in the interface. Taken together, our results show that the p52/Tfb2 subunit of TFIIH regulates the function of XPB through pair-wise interactions as described previously for p44 and XPD.

The nuclear factor y (NF-Y) trimer and TFIID contain histone fold subunits, and their binding to the CCAAT and Initiator elements of the major histocompatibility complex class II Ea promoter is required for transcriptional activation. Using agarose-electrophoretic mobility shift assay we found that NF-Y increases the affinity of holo-TFIID for Ea in a CCAAT- and Inr-dependent manner. We began to dissect the interplay between NF-Y- and TBP-associated factors PO1II (TAF(II)s)-containing histone fold domains in protein-protein interactions and transfections. hTAF(II)20, hTAF(II)28, and hTAF(II)18-hTAF(II)28 bind to the NF-Y B-NF-YC histone fold dimer; hTAF(II)80 and hTAF(II)31-hTAF(II)80 interact with the trimer but not with the NF-YB-NF-YC dimer. The histone fold alpha2 helix of hTAF(II)80 is not required for NF-Y association, as determined by interactions with the naturally occurring splice variant hTAF(II)80 delta. Expression of hTAF(II)28 and hTAF(II)18 in mouse cells significantly and specifically reduced NF-Y activation in GAL4-based experiments, whereas hTAF(II)20 and hTAF(II)135 increased it. These results indicate that NF-Y (i) recruits purified holo-TFIID in vitro and (ii) can associate multiple TAF(II)s, potentially accommodating different core promoter architectures.

The major postembryonic developmental events happening in insect life, including molting and metamorphosis, are regulated and coordinated temporally by pulses of ecdysone. The biological activity of this steroid hormone is mediated by two nuclear receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein (USP). The crystal structure of the ligand-binding domain from the lepidopteran Heliothis virescens USP reported here shows that the loop connecting helices H1 and H3 precludes the canonical agonist conformation. The key residues that stabilize this unique loop conformation are strictly conserved within the lepidopteran USP family. The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of this receptor provides the template for designing inhibitors, which could be utilized as a novel type of environmentally safe insecticides.

5'-Nucleotidases are the catabolic members of the substrate cycles postulated to be involved in the regulation of intracellular deoxyribonucleoside triphosphate pools. Here, we attempt to identify the nature of the nucleotidases. Earlier, we constructed various mammalian cell lines that can be induced to overproduce the high K(m) 5'-nucleotidase (hkm-NT) or the 5'(3')-deoxynucleotidase (dNT-1). Now we labeled control and induced human 293 cells and hamster V79 cells with radioactive hypoxanthine or uridine and during a chase measured quantitatively the metabolism of ribo- and deoxyribonucleotides, DNA replication, and excretion of nucleosides into the medium. Overproduction of hkm-NT greatly increased excretion of inosine and guanosine but did not affect adenosine or deoxyribonucleosides. dNT-1 overproduction increased excretion of deoxycytidine, thymidine, and in particular deoxyuridine but also uridine and cytidine. We conclude that the hkm-NT is not involved in the regulation of deoxyribonucleotide pools but affects IMP and GTP pools. dNT-1, instead, appears to be the catabolic arm of substrate cycles regulating pyrimidine nucleotide pools.

Androgens, like progestins, are 3-ketosteroids with structural differences restricted to the 17β substituent in the steroid D-ring. To better understand the specific recognition of ligands by the human androgen receptor (hAR), a homology model of the ligand-binding domain (LBD) was constructed based on the progesterone receptor LBD crystal structure. Several mutants of residues potentially involved in the specific recognition of ligands in the hAR were constructed and tested for their ability to bind agonists. Their transactivation capacity in response to agonist (R1881) and antagonists (cyproterone acetate, hydroxyflutamide, and ICI 176344) was also measured. Substitution of His874 by alanine, only marginally impairs the ligand-binding and transactivation capacity of the hAR receptor. In contrast, mutations of Thr877 and, to a greater extent, Asn705 perturb ligand recognition, alter transactivation efficiency, and broaden receptor specificity. Interestingly, the N705A mutant acquires progesterone receptor (PR) properties for agonist ligands but, unlike wild type AR and PR, loses the capacity to repress transactivation with nonsteroidal antagonists. Models of the hAR·LBD complexes with several ligands are presented, which suggests new directions for drug design.

DNA-gyrase exhibits an unusual ATP-binding site that is formed as a result of gyrase B subunit dimerization, a structural transition that is also essential for DNA capture during the topoisomerization cycle. Previous structural studies on Escherichia coli DNA-gyrase B revealed that dimerization is the result of a polypeptidic exchange involving the N-terminal 14 amino acids. To provide experimental data that dimerization is critical for ATPase activity and enzyme turnover, we generated mutants with reduced dimerization by mutating the two most conserved residues of the GyrB N-terminal arm (Tyr-5 and Ile-10 residues). Our data demonstrate that the hydrophobic Ile-10 residue plays an important role in enzyme dimerization and the nucleotide-protein contact mediated by Tyr-5 side chain residue helps the dimerization process. Analysis of ATPase activities of mutant proteins provides evidence that dimerization enhances the ATP-hydrolysis turnover. The structure of the Y5S mutant of the N-terminal 43-kDa fragment of E. coli DNA GyrB subunit indicates that Tyr-5 residue provides a scaffold for the ATP-hydrolysis center. We describe a channel formed at the dimer interface that provides a structural mechanism to allow reactive water molecules to access the gamma-phosphate group of the bound ATP molecule. Together, these results demonstrate that dimerization strongly contributes to the folding and stability of the catalytic site for ATP hydrolysis. A role for the essential Mg(2+) ion for the orientation of the phosphate groups of the bound nucleotide inside the reactive pocket was also uncovered by superposition of the 5'-adenylyl beta-gamma-imidodiphosphate (ADPNP) wild-type structure to the salt-free ADPNP structure.

A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant. The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent content is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution.

The amino acid sequences of pig muscle and of yeast glyceraldehyde-3-phosphate dehydrogenase are compared with the three-dimensional structure of the lobster muscle enzyme. Residues in sheet and helical regions, on the exterior and interior, in subunit and domain interfaces, as well as residues in the active site have been examined for evolutionary conservation. The residues in the first (NAD binding) domain (1-147) are less conserved than residues in the second (catalytic) domain (148-334) probably because there are fewer internal residues and fewer residues involved in interactions between subunits. Residues in subunit interface are conserved to a significantly greater extent than others, and those involved in catalysis are conserved most of all. Patterns of residues in helices and sheets follow those found for other proteins.